The Factor VII (FVII) is a glycoprotein depending on vitamin K, which is involved in the extrinsic route of blood coagulation. In its activated form (FVIIa), the factor VIIa is involved in the coagulation process by forming a complex with the tissue factor (TF) and by activating the factor X and the factor IX into factor Xa and IXa, respectively. So FVIIa has the capability of triggering coagulation of blood when the cascade of reactions leading to blood coagulation is interrupted or deficient, for example in the absence of factor VII or IX. This is why the factor VIIa has been used for a long time as a drug for treating certain disorders of blood coagulation expressed by bleeding, and notably for treating patients having factor VIII deficiency (hemophilia of type A) or factor IX deficiency (hemophilia of type B) and having inhibitors against these factors, for treating patients having congenital factor VII deficiency, or as a product for preventing hemorrhages which may occur during surgical operations.
The factor VII is secreted in the form of a single peptide chain of 406 amino acids, with a molecular weight of about 50 kDa, which is cut at the Arg152-Ile153 (Arginine 152-Isoleucine 153) bond during its activation into FVIIa. The Factor VIIa resulting from this therefore consists of a light chain of 152 amino acids, with a molecular weight of about 20 kDa, and of a heavy chain of 254 amino acids, with a molecular weight of about 30 kDa, bound together through a single disulfide bridge (Cys135-Cys262).
Four distinct structural domains may be identified in the sequence of the factor VII: an N-terminal γ-carboxylic domain (Gla domain), two “epidermal growth factor (EGF)-like”: domains as well as a serine protease domain.
The plasma Factor VIIa moreover includes several post-translational modifications among which: γ-carboxylation of the first ten glutamic acids of its sequence, partial hydroxylation of the aspartic acid 63, O-glycosylation of the serines 52 and 60 (Ser52 and Ser60) and N-glycosylation of the asparagines 145 and 322 (Asn145 and Asn322) (Fenaille F. et al., Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII. Glycoconj. J. 25.9 (2008): 827-42).
The biological activity of the factor VII thus strongly depends on the nature and on the proportion of the oligosaccharide structures which are attached to the protein, and which may affect many aspects of the therapeutic efficiency such as for example, solubility, resistance to proteolytic attacks, thermal inactivation, immunogenicity, half-life, bioactivity, bioavailability and stability of the factor VII.
It appears that these post-translational modifications, which often vary from one plasma factor VII molecule to another, lead to some heterogeneity of the molecules present in the pharmaceutical compositions of factor VII. Such heterogeneity notably has the drawback of complicating the steps for formulating the factor VII with view to the preparation of pharmaceutical compositions intended for treating patients. Indeed, it is frequent that a significant proportion of the purified factor VII molecules precipitate during the formulation step: the result of this is a loss of active raw material, as well as the presence of the factor VII having become inactive into pharmaceutical compositions intended for therapeutic treatment.
Because of the numerous drawbacks of the use of human plasma (risks of viral contamination, difficulty in purification, supply . . . ) as a source of pharmaceutical products, it is now preferred to produce the factor VII in recombinant or transgenic systems. However, insofar that glycosylation is a complex post-translational modification which directly depends on the cell system used, large scale production of proteins in heterologous cells often leads to the production of polypeptides having an identical primary structure, but having variable oligosaccharide structures.
For example, this is the case of NovoSeven®, a drug which has been authorized on the European market since 1996 and authorized on the American market in 1999, produced by the Danish firm NovoNordisk, the active ingredient of which is eptacog alfa (human recombinant activated coagulation Factor VII produced by genetic engineering from BKH kidney cells of newborn hamsters).
This is also the case, of recombinant transgenic FVII described in application EP 2 037 955 A, filed on May 31, 2007 by LFB Biotechnologies.
It thus appears that for these recombinant factors VII, the heterogeneity of the post-translational modifications borne by the molecules of Factor VII perturb the formulation step and contribute to reducing the stability and/or the specific activity of the final pharmaceutical products, by promoting the presence of precipitated factor VII molecules. The result of this is many difficulties for managing to prepare pharmaceutically acceptable compositions having a uniform and predetermined clinical efficiency.
Therefore there exists an increasing need for factor VII compositions having improved chemical and physical stability at room temperature and capable of facilitating and improving the yield of the formulation steps aiming at preparing pharmaceutical compositions intended for the treatment of patients affected by hemophilia with inhibitors or congenital factor VII deficiency.